Regulation of gene function occurs by several mechanisms in eukaryotic cells. Amongst these mechanisms are gene transcription regulation, mRNA translation regulation, and post-translation modification of proteins. The term “post-translation modification of proteins” includes several processes whereby proteins are structurally modified which may result in alterations in cellular, sub-cellular localization, stability, transport, interaction specificity, enzymatic activity, and numerous other characteristics.
Common and extensively studied post-translational modification processes include acetylation, glycosylation, and phosphorylation. Less well characterized is a process that involves the covalent addition of polymers of ADP-ribose to protein targets. The polymer is termed Poly (ADP-ribose) and the enzyme(s) responsible for this activity have been variously called Poly(ADP-ribose) Polymerase (PARP1), Poly(ADP-ribose) Synthetase (PARS), or ADP-Ribosyl Transferase (ADPRT), hereinafter called “PARP1”. PARP1 is an enzyme located in the nuclei of cells of various organs, including muscle, heart and brain cells. PARP1 plays a physiological role in the repair of strand breaks in DNA. Once activated by damaged DNA fragments, PARP1 catalyzes the attachment of up to 100 ADP-ribose units to a variety of nuclear proteins, including histones and PARP1 itself. While the exact range of functions of PARP1 has not been fully established, this enzyme is thought to play a role in enhancing DNA repair. At least three members of a structurally related set of gene products have been shown to catalyze poly-ADP-ribosylation. Currently, the most studied member of this gene family is PARP1. The PARP1 gene product is expressed at high levels in the nuclei of cells and is dependent upon DNA damage for activation. Without being bound by any theory, it is believed that PARP1 binds to DNA single or double stranded breaks through an amino terminal DNA binding domain. The binding activates the carboxy terminal catalytic domain and results in the formation of polymers of ADP-ribose on target molecules. PARP1 is itself a target of poly ADP-ribosylation by virtue of a centrally located automodification domain. The ribosylation of PARP1 causes dissociation of the PARP1 molecules from the DNA. The entire process of binding, ribosylation, and dissociation occurs very rapidly. It has been suggested that this transient binding of PARP1 to sites of DNA damage result in the recruitment of DNA repair machinery or may act to suppress to recombination long enough for the recruitment of repair machinery.
The source of ADP-ribose for the PARP reaction is nicotinamide adenosine dinucleotide (NAD). NAD is synthesized in cells from cellular ATP stores and thus high levels of activation of PARP activity can rapidly lead to depletion of cellular energy stores. It has been demonstrated that induction of PARP activity can lead to cell death that is correlated with depletion of cellular NAD and ATP pools. PARP activity is induced in many instances of oxidative stress or during inflammation. For example, during reperfusion of ischemic tissues reactive nitric oxide is generated and nitric oxide results in the generation of additional reactive oxygen species including hydrogen peroxide, peroxynitrate and hydroxyl radical. These latter species can directly damage DNA and the resulting damage induces activation of PARP activity. Frequently, it appears that sufficient activation of PARP activity occurs such that the cellular energy stores are depleted and the cell dies. A similar mechanism is believe to operate during inflammation when endothelial cells and pro-inflammatory cells synthesize nitric oxide which results in oxidative DNA damage in surrounding cells and the subsequent activation of PARP activity. The cell death that results from PARP activation is believed to be a major contributing factor in the extent of tissue damage that results from ischemia-reperfusion injury or from inflammation.
Two lines of evidence suggest that PARP activity is a critical element in those processes. First, chemical inhibitors of PARP activity have been successfully utilized to reduce tissue damage resulting in animal models of ischemia-reperfusion injury or inflammation. Secondly, mice in which both alleles of PARP1 have been disabled (PARP1 knockout mice or PARP1 mutant mice) are resistant to numerous forms of ischemia-reperfusion injury and detrimental effects of inflammation.
Inhibition of PARP activity has also been shown to be potentially useful in the treatment of human cancer. PARP small molecule inhibitors sensitize treated tumor cell lines to killing by ionizing radiation and by some DNA damaging chemotherapeutic drugs. While the PARP inhibitors by themselves generally do not have significant anti-tumor effect, when combined with a chemotherapeutic they can induce tumor regression at concentrations of the chemotherapeutic that are ineffective by themselves. Further, PARP1 mutant mice and PARP1 mutant cell lines are sensitive to radiation and similar types of chemotherapeutic drugs.
Currently known PARP inhibiting compounds are not approved for clinical use in treating a variety of diseases. Therefore, there is a need for PARP inhibitors which are clinically useful.